Compositions and methods for treatment of hepatitis C virus-associated diseases

ABSTRACT

Antisense oligonucleotides are provided which are complementary to and hybridizable with at least a portion of HCV RNA and which are capable of inhibiting the function of the HCV RNA. These oligonucleotides can be administered to inhibit the activity of Hepatitis C virus in vivo or in vitro. These compounds can be used either prophylactically or therapeutically to reduce the severity of diseases associated with Hepatitis C virus, and for diagnosis and detection of HCV and HCV-associated diseases. Methods of using these compounds are also disclosed.

INTRODUCTION

[0001] This application is a continuation-in-part of U.S. Ser. No.09/690,936 filed Oct. 18, 2000, which is a continuation of U.S. Ser. No.08/988,321, filed Dec. 10, 1997, which is a continuation-in-part of U.S.Ser. No. 08/650,093, filed May 17, 1996, which is a continuation-in-partof U.S. Ser. No. 08/452,841, filed May 30, 1995, which in turn is acontinuation-in-part of U.S. Ser. No. 08/397,220, filed Mar. 9, 1995,which is a continuation-in-part of U.S. Ser. No. 07/945,289, filed Sep.10, 1992.

FIELD OF THE INVENTION

[0002] This invention relates to the design and synthesis of antisenseoligonucleotides which can be administered to inhibit the activity ofHepatitis C virus in vivo or in vitro and to prevent or treat HepatitisC virus-associated disease. These compounds can be used eitherprophylactically or therapeutically to reduce the severity of diseasesassociated with Hepatitis C virus. These compounds can also be used fordetection of Hepatitis C virus and diagnosis of Hepatitis Cvirus-associated diseases. Oligonucleotides which are specificallyhybridizable with Hepatitis C virus RNA targets and are capable ofinhibiting the function of these RNA targets are disclosed. Methods ofusing these compounds are also disclosed.

BACKGROUND OF THE INVENTION

[0003] The predominant form of hepatitis currently resulting fromtransfusions is not related to the previously characterized Hepatitis Avirus or Hepatitis B virus and has, consequently, been referred to asNon-A, Non-B Hepatitis (NANBH). NANBH currently accounts for over 90% ofcases of post-transfusion hepatitis. Estimates of the frequency of NANBHin transfusion recipients range from 5%-13% for those receivingvolunteer blood, or 25-54% for those receiving blood from commercialsources.

[0004] Acute NANBH, while often less severe than acute disease caused byHepatitis A or Hepatitis B viruses, can lead to severe or fulminanthepatitis. Of greater concern, progression to chronic hepatitis is muchmore common after NANBH than after either Hepatitis A or Hepatitis Binfection.

[0005] Chronic NANBH has been reported in 10%-70% of infectedindividuals. This form of hepatitis can be transmitted even byasymptomatic patients, and frequently progresses to malignant diseasesuch as cirrhosis and hepatocellular carcinoma. Chronic activehepatitis, with or without cirrhosis, is seen in 44%-90% ofposttransfusion hepatitis cases. Of those patients who developedcirrhosis, approximately one-fourth died of liver failure.

[0006] Chronic active NANBH is a significant problem to hemophiliacs whoare dependent on blood products; 5%-11% of hemophiliacs die of chronicend-stage liver disease. Cases of NANBH other than those traceable toblood or blood products are frequently associated with hospitalexposure, accidental needle stick, or tattooing. Transmission throughclose personal contact also occurs, though this is less common for NANBHthan for Hepatitis B.

[0007] The causative agent of the majority of NANBH has been identifiedand is now referred to as Hepatitis C Virus (HCV). Houghton et al., EPPublication 318,216; Choo et al., Science 1989, 244, 359-362. Based onserological studies using recombinant DNA-generated antigens it is nowclear that HCV is the causative agent of most cases of post-transfusionNANBH. The HCV genome is a positive or plus-strand RNA genome. EPPublication 318,216 (Houghton et al.) discloses partial genomicsequences of HCV-1, and teaches recombinant DNA methods of cloning andexpressing HCV sequences and HCV polypeptides, techniques of HCVimmunodiagnostics, HCV probe diagnostic techniques, anti-HCV antibodies,and methods of isolating new HCV sequences. Houghton et al. alsodisclose additional HCV sequences and teach application of thesesequences and polypeptides in immunodiagnostics, probe diagnostics,anti-HCV antibody production, PCR technology and recombinant DNAtechnology. The concept of using antisense polynucleotides as inhibitorsof viral replication is disclosed, but no specific targets are taught.Oligomer probes and primers based on the sequences disclosed are alsoprovided. EP Publication 419,182 (Miyamura et al.) discloses new HCVisolates J1 and J7 and use of sequences distinct from HCV-1 sequencesfor screens and diagnostics.

[0008] The only treatment regimen shown to be effective for thetreatment of chronic NANBH is interferon-α. Most NANBH patients show animprovement of clinical symptoms during interferon treatment, butrelapse is observed in at least half of patients when treatment isinterrupted. Long term remissions are achieved in only about 20% ofpatients even after 6 months of therapy. Significant improvements inantiviral therapy are therefore greatly desired. An obvious need existsfor a clinically effective antiviral therapy for acute and chronic HCVinfections. Such an antiviral would also be useful for preventing thedevelopment of HCV-associated disease, for example for individualsaccidently exposed to blood products containing infectious HCV. There isalso a need for research reagents and diagnostics which are able todifferentiate HCV-derived hepatitis from hepatitis caused by otheragents and which are therefore useful in designing appropriatetherapeutic regimes.

[0009] Antisense Oligonucleotides

[0010] Oligonucleotides are commonly used as research reagents anddiagnostics. For example, antisense oligonucleotides, which, by nature,are able to inhibit gene expression with exquisite specificity, areoften used by those of ordinary skill to elucidate the function ofparticular genes, for example to determine which viral genes areessential for replication, or to distinguish between the functions ofvarious members of a biological pathway. This specific inhibitory effecthas, therefore, been exploited for research use. This specificity andsensitivity is also harnessed by those of skill in the art fordiagnostic uses. Viruses capable of causing similar hepatic symptoms canbe easily and readily distinguished in patient samples, allowing propertreatment to be implemented. Antisense oligonucleotide inhibition ofviral activity in vitro is useful as a means to determine a propercourse of therapeutic treatment. For example, before a patient suspectedof having an HCV infection is contacted with an oligonucleotidecomposition of the present invention, cells, tissues or a bodily fluidfrom the patient can be contacted with the oligonucleotide andinhibition of viral RNA function can be assayed. Effective in vitroinhibition of HCV RNA function, routinely assayable by methods such asNorthern blot or RT-PCR to measure RNA replication, or Western blot orELISA to measure protein translation, indicates that the infection willbe responsive to the oligonucleotide treatment.

[0011] Oligonucleotides have also been employed as therapeutic moietiesin the treatment of disease states in animals and man. For example,workers in the field have now identified antisense, triplex and otheroligonucleotide compositions which are capable of modulating expressionof genes implicated in viral, fungal and metabolic diseases. Asexamples, U.S. Pat. No. 5,166,195 issued Nov. 24, 1992, providesoligonucleotide inhibitors of HIV. U.S. Pat. No. 5,004,810, issued Apr.2, 1991, provides oligomers capable of hybridizing to herpes simplexvirus Vmw65 mRNA and inhibiting replication. U.S. Pat. No. 5,194,428,issued Mar. 16, 1993, provides antisense oligonucleotides havingantiviral activity against influenzavirus. U.S. Pat. No. 4,806,463,issued Feb. 21, 1989, provides antisense oligonucleotides and methodsusing them to inhibit HTLV-III replication. U.S. Pat. No. 5,276,019 andU.S. Pat. No. 5,264,423 (Cohen et al.) are directed to phosphorothioateoligonucleotide analogs used to prevent replication of foreign nucleicacids in cells. Antisense oligonucleotides have been safely andeffectively administered to humans and clinical trials of severalantisense oligonucleotide drugs are presently underway. Thephosphorothioate oligonucleotide, ISIS 2922, has been shown to beeffective against cytomegalovirus retinitis in AIDS patients. BioWorldToday, Apr. 29, 1994, p. 3. It is thus established that oligonucleotidescan be useful drugs for treatment of cells and animal subjects,especially humans.

[0012] Seki et al. have disclosed antisense compounds complementary tospecific defined regions of the HCV genome. Canadian patent application2,104,649.

[0013] Hang et al. have disclosed antisense oligonucleotidescomplementary to the 5′ untranslated region of HCV for controllingtranslation of HCV proteins, and methods of using them. WO 94/08002.

[0014] Blum et al. have disclosed antisense oligonucleotidescomplementary to an RNA complementary to a portion of a hepatitis viralgenome which encodes the terminal protein region of the viralpolymerase, and methods of inhibiting replication of a hepatitis virususing such oligonucleotides. WO 94/24864.

[0015] Wakita and Wands have used sense and antisense oligonucleotidesto determine the role of the 5′ end untranslated region in the lifecycle of HCV. Antisense oligonucleotides targeted to three regions ofthe 5′ untranslated region and one region of the core protein codingregion effectively blocked in vitro translation of HCV protein,suggesting that these domains may be critical for HCV translation. J.Biol. Chem. 1994, 269, 14205-14210.

SUMMARY OF THE INVENTION

[0016] In accordance with the present invention, compositions andmethods for modulating the effects of HCV infection are provided.Oligonucleotides which are complementary to, and specificallyhybridizable with, selected sequences of HCV RNA and which are capableof inhibiting the function of the HCV RNA are provided. The HCVpolyprotein translation initiation codon region is a preferred target.An oligonucleotide (SEQ ID NO: 6) targeted to nucleotides 330-349 of theinitiation codon region is particularly preferred, and this sequencecomprising a 5-methylcytidine at every cytidine residue is even morepreferred. Methods for diagnosing or treating disease states byadministering oligonucleotides, either alone or in combination with apharmaceutically acceptable carrier, to animals suspected of havingHCV-associated diseases are also provided.

DETAILED DESCRIPTION OF THE INVENTION

[0017] Several regions of the HCV genome have been identified asantisense targets in the present invention. The size of the HCV genomeis approximately 9400 nucleotides, with a single translational readingframe encoding a polyprotein which is subsequently processed to severalstructural and non-structural proteins. It should be noted that sequenceavailability and nucleotide numbering schemes vary from strain tostrain. The 5′ untranslated region (5′ UTR) or 5′ noncoding region (5′NCR) of HCV consists of approximately 341 nucleotides upstream of thepolyprotein translation initiation codon. A hairpin loop present atnucleotides 1-22 at the 5′ end of the genome (HCV-1) identified hereinas the “5′ end hairpin loop” is believed to serve as a recognitionsignal for the viral replicase or nucleocapsid proteins. Han et al.,Proc. Natl. Acad. Sci. 1991, 88, 1711-1715. The 540 untranslated regionis believed to have a secondary structure which includes six stem-loopstructures, designated loops A-F. Loop A is present at approximatelynucleotides 13-50, loop B at approximately nucleotides 51-88, loop C atapproximately nucleotides 100-120, loop D at approximately nucleotides147-162, loop E at approximately nucleotides 163-217, and loop F atapproximately nucleotides 218-307. Tsukiyama-Kohara et al., J. Virol.1992, 66, 1476-1483. These structures are well conserved between the twomajor HCV groups.

[0018] Three small (12-16 amino acids each) open reading frames (ORFs)are located in the 5′-untranslated region of HCV RNA. These ORFs may beinvolved in control of translation. The ORF 3 translation initiationcodon as denominated herein is found at nucleotides 315-317 of HCV-1according to the scheme of Han et al., Proc. Natl. Acad. Sci. 1991, 88,1711-1715; and at nucleotides −127 to −125 according to the scheme ofChoo et al., Proc. Natl. Acad. Sci. 1991, 88, 2451-2455.

[0019] The polyprotein translation initiation codon as denominatedherein is an AUG sequence located at nucleotides 342-344 of HCV-1according to Han et al., Proc. Natl. Acad. Sci. 1991, 88, 1711-1715 orat nucleotide 1-3 according to the HCV-1 numbering scheme of Choo etal., Proc. Natl. Acad. Sci. 1991, 88, 2451-2455. Extending downstream(toward 3′ end) from the polyprotein AUG is the core protein codingregion.

[0020] The 3′ untranslated region, as denominated herein, consists ofnucleotides downstream of the polyprotein translation termination site(ending at nt 9037 according to Choo et al.; nt 9377 according toschemes of Han and Inchauspe). Nucleotides 9697-9716 (numbering schemeof Inchauspe for HCV-H) at the 3′ terminus of the genome within the 31untranslated region can be organized into a stable hairpin loopstructure identified herein as the 3′ hairpin loop. A short nucleotidestretch (R2) immediately upstream (nt 9691-9696 of HCV-H) of the 3′hairpin, and denominated herein “the R2 sequence”, is thought to play arole in cyclization of the viral RNA, possibly in combination with a setof 5′ end 6-base-pair repeats of the same sequence at nt 23-28 and38-43. (Inchauspe et al., Proc. Natl. Acad. Sci. 1991, 88, 10292-10296)is identified herein as “5′ end 6-base-pair repeat”. Palindromesequences present near the 3′ end of the genome (nucleotides 9312-9342according to the scheme of Takamizawa et al., J. Virol. 1991, 65,1105-1113) are capable of forming a stable secondary structure. This isreferred to herein as the 340 end palindrome region.

[0021] Antisense Oligonucleotides

[0022] The present invention employs oligonucleotides 5 to 50nucleotides in length which are specifically hybridizable with hepatitisC virus RNA and are capable of inhibiting the function of the HCV RNA.In preferred embodiments, oligonucleotides are targeted to the 5′ endhairpin loop, 5′ end 6-base-pair repeats, 5′ end untranslated region,polyprotein translation initiation codon, core protein coding region,ORF 3 translation initiation codon, 3′-untranslated region, 3′ endpalindrome region, R2 sequence and 3′ end hairpin loop region of HCVRNA. This relationship between an oligonucleotide and the nucleic acidsequence to which it is targeted is commonly referred to as “antisense”.“Targeting” an oligonucleotide to a chosen nucleic acid target, in thecontext of this invention, is a multistep process. The process usuallybegins with identifying a nucleic acid sequence whose function is to bemodulated. This may be, as examples, a cellular gene (or mRNA made fromthe gene) whose expression is associated with a particular diseasestate, or a foreign nucleic acid (RNA or DNA) from an infectious agent.In the present invention, the target is the 51 end hairpin loop, 5′ end6-base-pair repeats, ORF 3 translation initiation codon (all of whichare contained within the 5′ UTR), polyprotein translation initiationcodon, core protein coding region (both of which are contained withinthe coding region), 3′ end palindrome region, R2 sequence or 3′ endhairpin loop (all of which are contained within the 3′ UTR) of HCV RNA.The targeting process also includes determination of a site or siteswithin the nucleic acid sequence for the oligonucleotide interaction tooccur such that the desired effect, i.e., inhibition of HCV RNAfunction, will result. Once the target site or sites have beenidentified, oligonucleotides are chosen which are sufficientlycomplementary to the target, i.e., hybridize sufficiently well and withsufficient specificity, to give the desired modulation.

[0023] In the context of this invention “modulation” means eitherinhibition or stimulation. Inhibition of HCV RNA function is presentlythe preferred form of modulation in the present invention. Theoligonucleotides are able to inhibit the function of viral RNA byinterfering with its replication, transcription into mRNA, translationinto protein, packaging into viral particles or any other activitynecessary to its overall biological function. The failure of the RNA toperform all or part of its function results in failure of all or aportion of the normal life cycle of the virus. This inhibition can bemeasured, in samples derived from either in vitro or in vivo (animal)systems, in ways which are routine in the art, for example by RT-PCR orNorthern blot assay of HCV RNA levels or by in vitro translation,Western blot or ELISA assay of protein expression as taught in theexamples of the instant application. “Hybridization”, in the context ofthis invention, means hydrogen bonding, also known as Watson-Crick basepairing, between complementary bases, usually on opposite nucleic acidstrands or two regions of a nucleic acid strand. Guanine and cytosineare examples of complementary bases which are known to form threehydrogen bonds between them. Adenine and thymine are examples ofcomplementary bases which form two hydrogen bonds between them.“Specifically hybridizable” and “complementary” are terms which are usedto indicate a sufficient degree of complementarity such that stable andspecific binding occurs between the DNA or RNA target and theoligonucleotide. It is understood that an oligonucleotide need not be100% complementary to its target nucleic acid sequence to bespecifically hybridizable. An oligonucleotide is specificallyhybridizable when binding of the oligonucleotide to the targetinterferes with the normal function of the target molecule to cause aloss of utility, and there is a sufficient degree of complementarity toavoid non-specific binding of the oligonucleotide to non-targetsequences under conditions in which specific binding is desired, i.e.,under physiological conditions in the case of in vivo assays ortherapeutic treatment or, in the case of in vitro assays, underconditions in which the assays are conducted.

[0024] In the context of this invention, the term “oligonucleotide”refers to an oligomer or polymer of nucleotide or nucleoside monomersconsisting of naturally occurring bases, sugars and intersugar(backbone) linkages. The term “oligonucleotide” also includes oligomersor polymers comprising non-naturally occurring monomers, or portionsthereof, which function similarly. Such modified or substitutedoligonucleotides are often preferred over native forms because ofproperties such as, for example, enhanced cellular uptake, increasedstability in the presence of nucleases, or enhanced target affinity. Anumber of nucleotide and nucleoside modifications have been shown tomake the oligonucleotide into which they are incorporated more resistantto nuclease digestion than the native oligodeoxynucleotide. Nucleaseresistance is routinely measured by incubating oligonucleotides withcellular extracts or isolated nuclease solutions and measuring theextent of intact oligonucleotide remaining over time, usually by gelelectrophoresis. Oligonucleotides which have been modified to enhancetheir nuclease resistance survive intact for a longer time thanunmodified oligonucleotides. A number of modifications have also beenshown to increase binding (affinity) of the oligonucleotide to itstarget. Affinity of an oligonucleotide for its target is routinelydetermined by measuring the Tm of an oligonucleotide/target pair, whichis the temperature at which the oligonucleotide and target dissociate.Dissociation is detected spectrophotometrically. The higher the Tm, thegreater the affinity of the oligonucleotide for the target. In somecases, oligonucleotide modifications which enhance target bindingaffinity are also, independently, able to enhance nuclease resistance.

[0025] Specific examples of some preferred oligonucleotides envisionedfor this invention may contain phosphorothioates (P═S),phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkylintersugar linkages or short chain heteroatomic or heterocyclicintersugar (“backbone”) linkages at one or more positions instead of thenative phosphodiester (P═O) backbone. Most preferred arephosphorothioates and those with CH₂—NH—O—CH₂, CH₂—N(CH₃)—O—CH₂ [knownas a methylene(methylimino) or MMI backbone], CH₂—O—N(CH₃)—CH₂,CH₂—N(CH₃) —N(CH₃) —CH₂ and O—N(CH₃) —CH₂—CH₂ backbones (wherephosphodiester is O—P—O—CH₂). Also preferred are oligonucleotides havingmorpholino backbone structures. Summerton, J. E. and Weller, D. D., U.S.Pat. No. 5,034,506. In other preferred embodiments, such as theprotein-nucleic acid or peptide-nucleic acid (PNA) backbone, thephosphodiester backbone of the oligonucleotide may be replaced with apolyamide backbone, the bases being bound directly or indirectly to theaza nitrogen atoms of the polyamide backbone. P. E. Nielsen, M. Egholm,R. H. Berg, O. Buchardt, Science 1991, 254, 1497. Oligonucleotidescontaining one or more PNA, MMI or P═S backbone linkages are presentlymore preferred. Other preferred oligonucleotides may contain one or moresubstituted sugar moieties comprising one of the following at the 2′position: OH, SH, SCH₃, F, OCN, OCH₃OCH₃, OCH₃O(CH₂)_(n)CH₃,O(CH₂)_(n)NH₂ or O(CH₂)_(n)CH₃ where n is from 1 to about 10; C₁ to C₁₀lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl;Cl; Br; CN; CF₃; OCF₃; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; SOCH₃;SO₂CH₃; ONO₂; NO₂; N₃; NH₂; heterocycloalkyl; heterocycloalkaryl;aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleavinggroup; a cholesteryl group; a reporter group; an intercalator; a groupfor improving the pharmacokinetic properties of an oligonucleotide; or agroup for improving the pharmacodynamic properties of an oligonucleotideand other substituents having similar properties. Presently preferredmodifications include 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃), 2′-methoxy(2′-O—CH₃), 2′-propoxy (2′-OCH₂CH₂CH)₃ and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide and the 5′ position of 5′ terminal nucleotide.Oligonucleotides may also have sugar mimetics such as cyclobutyls inplace of the pentofuranosyl group.

[0026] The oligonucleotides of the invention may additionally oralternatively include nucleobase modifications or substitutions. As usedherein, “unmodified” or “natural” nucleobases include adenine (A),guanine (G), thymine (T), cytosine (C) and uracil (U). Modifiednucleobases known in the art include nucleobases found only infrequentlyor transiently in natural nucleic acids, e.g., hypoxanthine (whosecorresponding nucleotide, inosine, is sometimes referred to as a“universal base”), 6-methyladenine, 5-methylcytosine,5-hydroxymethylcytosine (HMC), glycosyl HMC and gentiobiosyl HMC, aswell synthetic nucleobases, e.g., 5-bromouracil, 5-hydroxymethyluracil,8-azaguanine, 7-deazaguanine, N⁶(6-aminohexyl)adenine and2,6-diaminopurine. Oligonucleotides in which cytosine bases are replacedby 5-methylcytosines are presently a preferred embodiment of theinvention.

[0027] Another preferred additional or alternative modification of theoligonucleotides of the invention involves chemically linking to theoligonucleotide one or more lipophilic moieties which enhance thecellular uptake of the oligonucleotide. Such lipophilic moieties may belinked to an oligonucleotide at several different positions on theoligonucleotide. Some preferred positions include the 3′ position of thesugar of the 3′ terminal nucleotide, the 5′ position of the sugar of the5′ terminal nucleotide, and the 2′ position of the sugar of anynucleotide. The N⁶ position of a purine nucleobase may also be utilizedto link a lipophilic moiety to an oligonucleotide of the invention. Suchlipophilic moieties known in the art include but are not limited to oneor more cholesteryl moieties, cholic acids, thioethers,thiocholesterols, aliphatic chains, e.g., dodecandiol or undecylresidues, phospholipids, polyamines or polyethylene glycol chains,adamantane acetic acid, palmityl moieties, octadecylamine orhexylamino-carbonyl-oxycholesterol moiety. Oligonucleotides comprisinglipophilic moieties, and methods for preparing such oligonucleotides, asdisclosed in U.S. Pat. No. 5,138,045, No. 5,218,105 and No. 5,459,255,the contents of which are hereby incorporated by reference.

[0028] Certain preferred oligonucleotides of this invention are chimericoligonucleotides. “Chimeric oligonucleotides” or “chimeras”, in thecontext of this invention, are oligonucleotides which contain two ormore chemically distinct regions, each made up of at least onenucleotide. These oligonucleotides typically contain at least one regionof modified nucleotides that confers one or more beneficial properties(such as, for example, increased nuclease resistance, increased uptakeinto cells, increased binding affinity for the RNA target) and a regionthat is a substrate for RNase H cleavage. In one preferred embodiment, achimeric oligonucleotide comprises at least one region modified toincrease target binding affinity, and, usually, a region that acts as asubstrate for RNAse H. Affinity of an oligonucleotide for its target (inthis case a nucleic acid encoding HCV RNA) is routinely determined bymeasuring the Tm of an oligonucleotide/target pair, which is thetemperature at which the oligonucleotide and target dissociate;dissociation is detected spectrophotometrically. The higher the Tm, thegreater the affinity of the oligonucleotide for the target. In a morepreferred embodiment, the region of the oligonucleotide which ismodified to increase HCV RNA binding affinity comprises at least onenucleotide modified at the 2′ position of the sugar, most preferably a2′-O-alkyl or 2′-fluoro-modified nucleotide. Such modifications areroutinely incorporated into oligonucleotides and these oligonucleotideshave been shown to have a higher Tm (i.e., higher target bindingaffinity) than 2′-deoxyoligonucleotides against a given target. Theeffect of such increased affinity is to greatly enhance antisenseoligonucleotide inhibition of HCV RNA function. RNAse H is a cellularendonuclease that cleaves the RNA strand of RNA:DNA duplexes; activationof this enzyme therefore results in cleavage of the RNA target, and thuscan greatly enhance the efficiency of antisense inhibition. Cleavage ofthe RNA target can be routinely demonstrated by gel electrophoresis. Inanother preferred embodiment, the chimeric oligonucleotide is alsomodified to enhance nuclease resistance. Cells contain a variety of exo-and endo-nucleases which can degrade nucleic acids. A number ofnucleotide and nucleoside modifications have been shown to make theoligonucleotide into which they are incorporated more resistant tonuclease digestion than the native oligodeoxynucleotide. Nucleaseresistance is routinely measured by incubating oligonucleotides withcellular extracts or isolated nuclease solutions and measuring theextent of intact oligonucleotide remaining over time, usually by gelelectrophoresis. Oligonucleotides which have been modified to enhancetheir nuclease resistance survive intact for a longer time thanunmodified oligonucleotides. A variety of oligonucleotide modificationshave been demonstrated to enhance or confer nuclease resistance. In somecases, oligonucleotide modifications which enhance target bindingaffinity are also, independently, able to enhance nuclease resistance.Oligonucleotides which contain at least one phosphorothioatemodification are presently more preferred.

[0029] The compounds of the present invention include bioequivalentcompounds, including pharmaceutically acceptable salts and prodrugs.

[0030] The compounds of the invention encompass any pharmaceuticallyacceptable salts, esters, or salts of such esters, or any other compoundwhich, upon administration to an animal including a human, is capable ofproviding (directly or indirectly) the biologically active metabolite orresidue thereof. Accordingly, for example, the disclosure is also drawnto pharmaceutically acceptable salts of the nucleic acids of theinvention and prodrugs of such nucleic acids.

[0031] Pharmaceutically acceptable salts are physiologically andpharmaceutically acceptable salts of the nucleic acids of the invention,i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto.

[0032] Pharmaceutically acceptable base addition salts are formed withmetals or amines, such as alkali and alkaline earth metals or organicamines. Examples of metals used as cations are sodium, potassium,magnesium, calcium, and the like. Examples of suitable amines areN,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine(see, for example, Berge et al., “Pharmaceutical Salts,” J. of PharmaSci. 1977, 66:1). The base addition salts of said acidic compounds areprepared by contacting the free acid form with a sufficient amount ofthe desired base to produce the salt in the conventional manner. Thefree acid form may be regenerated by contacting the salt form with anacid and isolating the free acid in the conventional manner. The freeacid forms differ from their respective salt forms somewhat in certainphysical properties such as solubility in polar solvents, but otherwisethe salts are equivalent to their respective free acid for purposes ofthe present invention. As used herein, a “pharmaceutical addition salt”includes a pharmaceutically acceptable salt of an acid form of one ofthe components of the compositions of the invention. These includeorganic or inorganic acid salts of the amines. Preferred acid salts arethe hydrochlorides, acetates, salicylates, nitrates and phosphates.Other suitable pharmaceutically acceptable salts are well known to thoseskilled in the art and include basic salts of a variety of inorganic andorganic acids, such as inorganic acids, for example hydrochloric acid,hydrobromic acid, sulfuric acid or phosphoric acid; with organiccarboxylic, sulfonic, sulfo or phospho acids or N-substituted sulfamicacids, for example acetic acid, propionic acid, glycolic acid, succinicacid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid,malic acid, tartaric acid, lactic acid, oxalic acid, gluconic acid,glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamicacid, mandelic acid, salicylic acid, 4-aminosalicylic acid,2-phenoxybenzoic acid, 2-acetoxybenzoic acid, nicotinic acid orisonicotinic acid; and with amino acids, such as the 20 alpha-aminoacids involved in the synthesis of proteins in nature, for exampleglutamic acid or aspartic acid, and also with phenylacetic acid,methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid,ethane-1,2-disulfonic acid, benzenesulfonic acid,4-methylbenzenesulfonic acid, naphthalene-2-sulfonic acid,naphthalene-1,5-disulfonic acid, 2- or 3-phosphoglycerate,glucose-6-phosphate, N-cyclohexylsulfamic acid (with the formation ofcyclamates), or with other acid organic compounds, such as ascorbicacid.

[0033] Pharmaceutically acceptable salts of compounds may also be formedwith a pharmaceutically acceptable cation. Suitable pharmaceuticallyacceptable cations are well known to those skilled in the art andinclude alkaline, alkaline earth, ammonium and quaternary ammoniumcations. Carbonates or hydrogen carbonates are also possible.

[0034] For oligonucleotides, examples of pharmaceutically acceptablesalts include but are not limited to (a) salts formed with cations suchas sodium, potassium, ammonium, magnesium, calcium, polyamines such asspermine and spermidine, etc.; (b) acid addition salts formed withinorganic acids, for example hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid and the like; (c) saltsformed with organic acids such as acetic acid, oxalic acid, tartaricacid, succinic acid, maleic acid, fumaric acid, gluconic acid, citricacid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmiticacid, alginic acid, polyglutamic acid, naphthalenesulfonic acid,methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonicacid, polygalacturonic acid, and the like; and (d) salts formed fromelemental anions such as chlorine, bromine, and iodine.

[0035] The oligonucleotides of the invention may additionally oralternatively be prepared to be delivered in a prodrug form. The term“prodrug” indicates a therapeutic agent that is prepared in an inactiveform that is converted to an active form (i.e., drug) within the body orcells thereof by the action of endogenous enzymes or other chemicalsand/or conditions. In particular, prodrug versions of theoligonucleotides of the invention are prepared as SATE[(S-acetyl-2-thioethyl)phosphate] derivatives according to the methodsdisclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993.

[0036] The oligonucleotides in accordance with this invention preferablyare from about 5 to about 50 nucleotides in length. In the context ofthis invention it is understood that this encompasses non-naturallyoccurring oligomers as hereinbefore described, having 5 to 50 monomers.

[0037] The oligonucleotides used in accordance with this invention maybe conveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including Applied Biosystems. Any other means for such synthesismay also be employed; the actual synthesis of the oligonucleotides iswell within the talents of the routineer. It is also well known to usesimilar techniques to prepare other oligonucleotides such as thephosphorothioates and alkylated derivatives. It is also well known touse similar techniques and commercially available modified amidites andcontrolled-pore glass (CPG) products such as those available from GlenResearch, Sterling, Va., to synthesize modified oligonucleotides such ascholesterol-modified oligonucleotides.

[0038] Methods of modulating the activity of HCV virus are provided, inwhich the virus, or cells, tissues or bodily fluid suspected ofcontaining the virus, is contacted with an oligonucleotide of theinvention. In the context of this invention, to “contact” means to addthe oligonucleotide to a preparation of the virus, or vice versa, or toadd the oligonucleotide to a preparation or isolate of cells, tissues orbodily fluid, or vice versa, or to add the oligonucleotide to virus,cells tissues or bodily fluid in situ, i.e., in an animal, especially ahuman.

[0039] The oligonucleotides of this invention can be used indiagnostics, therapeutics and as research reagents and kits. Since theoligonucleotides of this invention hybridize to RNA from HCV, sandwichand other assays can easily be constructed to exploit this fact.Provision of means for detecting hybridization of oligonucleotide withHCV or HCV RNA present in a sample suspected of containing it canroutinely be accomplished. Such provision may include enzymeconjugation, radiolabelling or any other suitable detection systems.Kits for detecting the presence or absence of HCV may also be prepared.The specific ability of the oligonucleotides of the invention to inhibitHCV RNA function can also be exploited in the detection and diagnosis ofHCV, HCV infection and HCV-associated diseases. As described in theexamples of the present application, the decrease in HCV RNA or proteinlevels as a result of oligonucleotide inhibition of HCV RNA function canbe routinely detected, for example by RT-PCR, Northern blot, Westernblot or ELISA.

[0040] For prophylactics and therapeutics, methods of preventingHCV-associated disease and of treating HCV infection and HCV-associateddisease are provided. The formulation of therapeutic compositions andtheir subsequent administration is believed to be within the skill inthe art. Oligonucleotides may be formulated in a pharmaceuticalcomposition, which may include carriers, thickeners, diluents, buffers,preservatives, surface active agents, liposomes or lipid formulationsand the like in addition to the oligonucleotide. Pharmaceuticalcompositions may also include one or more active ingredients such asinterferons, antimicrobial agents, anti-inflammatory agents,anesthetics, and the like. Formulations for parenteral administrationmay include sterile aqueous solutions which may also contain buffers,liposomes, diluents and other suitable additives.

[0041] The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic, vaginal, rectal,intranasal, epidermal and transdermal), oral or parenteral. Parenteraladministration includes intravenous drip, subcutaneous, intraperitonealor intramuscular injection, pulmonary administration, e.g., byinhalation or insufflation, or intracranial, e.g., intrathecal orintraventricular, administration. For oral administration, it has beenfound that oligonucleotides with at least one 2′-substitutedribonucleotide are particularly useful because of their absorption anddistribution characteristics. U.S. Pat. No. 5,591,721 issued to Agrawalet al. Oligonucleotides with at least one 2′-O-methoxyethyl modificationare believed to be particularly useful for oral administration.

[0042] Formulations for topical administration may include transdermalpatches, ointments, lotions, creams, gels, drops, suppositories, sprays,liquids and powders. Conventional pharmaceutical carriers, aqueous,powder or oily bases, thickeners and the like may be necessary ordesirable. Coated condoms, gloves and the like may also be useful.

[0043] Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable.

[0044] Compositions for parenteral administration may include sterileaqueous solutions which may also contain buffers, diluents and othersuitable additives.

[0045] Dosing is dependent on severity and responsiveness of thecondition to be treated, with course of treatment lasting from severaldays to several months or until a reduction in viral titer (routinelymeasured by Western blot, ELISA, RT-PCR, or RNA (Northern) blot, forexample) is effected or a diminution of disease state is achieved.Optimal dosing schedules are easily calculated from measurements of drugaccumulation in the body. Persons of ordinary skill can easily determineoptimum dosages, dosing methodologies and repetition rates.Therapeutically or prophylactically effective amounts (dosages) may varydepending on the relative potency of individual compositions, and cangenerally be routinely calculated based on molecular weight and EC50s inin vitro and/or animal studies. For example, given the molecular weightof drug compound (derived from oligonucleotide sequence and chemicalstructure) and an experimentally derived effective dose such as an IC₅₀,for example, a dose in mg/kg is routinely calculated. In general, dosageis from 0.001 μg to 100 g and may be administered once or several timesdaily, weekly, monthly or yearly, or even every 2 to 20 years.Pharmacokinetics of Antisense Oligonucleotides Because the primarypathology associated with HCV infection occurs in the liver of infectedindividuals, the ability of a potential anti-HCV compound to achievesignificant concentrations in the liver is advantageous. Pharmacokineticprofiles for a number of oligonucleotides, primarily phosphorothioateoligonucleotides, have been determined. Phosphorothioateoligonucleotides have been shown to have very similar pharmacokineticsand tissue distribution, regardless of sequence. This is characterizedin plasma by a rapid distribution phase (approximately 30 minutes) and aprolonged elimination phase (approximately 40 hours). Phosphorothioatesare found to be broadly distributed to peripheral tissues (i.e.,excepting the brain, which is reachable directly, e.g., byintraventricular drug administration, and in addition may be reachablevia a compromised blood-brain barrier in many nervous systemconditions), with the highest concentrations found in liver, renalcortex and bone marrow. There is good accumulation of intact compound inmost tissues, particularly liver, kidney and bone marrow, with veryextended compound half-life in tissues. Similar distribution profilesare found whether the oligonucleotide is administered intravenously orsubcutaneously. Furthermore, the pharmacokinetic and tissue distributionprofiles are very consistent among animal species, including rodents,monkeys and humans.

PREFERRED EMBODIMENTS OF THE INVENTION

[0046] It has been found that antisense oligonucleotides designed totarget viruses can be effective in diminishing viral infection.

[0047] In accordance with this invention, persons of ordinary skill inthe art will understand that messenger RNA includes not only thesequence information to encode a protein using the three letter geneticcode, but also associated ribonucleotides which form regions known tosuch persons as the 5′-untranslated region, the 3′-untranslated region,and the 5′ cap region, as well as ribonucleotides which form varioussecondary structures. Thus, oligonucleotides may be formulated inaccordance with this invention which are targeted wholly or in part tothese associated ribonucleotides as well as to the codingribonucleotides. In preferred embodiments, the oligonucleotide isspecifically hybridizable with the HCV 5′ end hairpin loop, 5′ end6-base-pair repeats, ORF 3 translation initiation codon, (all of whichare contained within the 5′ UTR) polyprotein translation initiationcodon, core protein coding region (both of which are contained withinthe coding region), R2 region, 3′ hairpin loop or 3′ end palindromeregion (all of which are contained within the 3′-untranslated region).It is to be expected that differences in the RNA of HCV from differentstrains and from different types within a strain exist. It is believedthat the regions of the various HCV strains serve essentially the samefunction for the respective strains and that interference withhomologous or analogous RNA regions will afford similar results in thevarious strains. This is believed to be so even though differences inthe nucleotide sequences among the strains exist.

[0048] Accordingly, nucleotide sequences set forth in the presentspecification will be understood to be representational for theparticular strain being described. Homologous or analogous sequences fordifferent strains of HCV are specifically contemplated as being withinthe scope of this invention. In preferred embodiments of the presentinvention, antisense oligonucleotides are targeted to the 5′untranslated region, core protein translation initiation codon region,core protein coding region, ORF 3 translation initiation codon and3′-untranslated region of HCV RNA.

[0049] In preferred embodiments, the antisense oligonucleotides arehybridizable with at least a portion of the polyprotein translationinitiation codon or with at least a portion of the core protein codingregion. The sequence of nucleotides 1-686 (SEQ ID NO: 37) comprises theentire 5′-untranslated region (nucleotides 1-341) and a 145-nucleotidecore region sequence of HCV RNA. A highly preferred oligonucleotidehybridizable with at least a portion of the polyprotein translationinitiation codon comprises SEQ ID NO: 6.

[0050] In vitro Evaluation of HCV Antisense Oligonucleotides

[0051] HCV replication in cell culture has not yet been achieved.Consequently, in vitro translation assays are used to evaluate antisenseoligonucleotides for anti-HCV activity. One such in vitro translationassay was used to evaluate oligonucleotide compounds for the ability toinhibit synthesis of HCV 5′ UTR-core-env transcript in a rabbitreticulocyte assay.

[0052] Cell-based assays are also used for evaluation ofoligonucleotides for anti-HCV activity. In one such assay, effects ofoligonucleotides on HCV RNA function are evaluated by measuring RNAand/or HCV core protein levels in transformed hepatocytes expressing the5′ end of the HCV genome. Recombinant HCV/vaccinia virus assays can alsobe used, such as those described in the examples of the presentapplication. Luciferase assays can be used, for example, as described inthe examples of the present application, in which recombinant vacciniavirus containing HCV sequences fused to luciferase sequences are used.Quantitation of luciferase with a luminometer is a simple way ofmeasuring HCV core protein expression and its inhibition by antisensecompounds. This can be done in cultured hepatocytes or in tissuesamples, such as liver biopsies, from treated animals.

[0053] Animal Models for HCV

[0054] There is no small animal model for chronic HCV infection. Arecombinant vaccinia/HCV/luciferase virus expression assay has beendeveloped for testing compounds in mice. Mice are inoculated withrecombinant vaccinia virus (either expressing HCV/luciferase orluciferase alone for a control). Organs (particularly liver) areharvested one or more days later and luciferase activity in the tissueis assayed by luminometry.

[0055] The following specific examples are provided for illustrativepurposes only and are not intended to limit the invention.

EXAMPLES Example 1 Oligonucleotide Synthesis

[0056] Unmodified oligodeoxynucleotides were synthesized on an automatedDNA synthesizer (Applied Biosystems model 380B) using standardphosphoramidite chemistry with oxidation by iodine.β-cyanoethyldiisopropyl-phosphoramidites were purchased from AppliedBiosystems (Foster City, Calif.). For phosphorothioate oligonucleotides,the standard oxidation bottle was replaced by a 0.2 M solution of³H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the stepwisethiation of the phosphite linkages. The thiation cycle wait step wasincreased to 68 seconds and was followed by the capping step.

[0057] 2′-methoxy oligonucleotides were synthesized using 2′-methoxyβ-cyanoethyldiisopropyl-phosphoramidites (Chemgenes, Needham Mass.) andthe standard cycle for unmodified oligonucleotides, except the wait stepafter pulse delivery of tetrazole and base was increased to 360 seconds.Other 2′-alkoxy oligonucleotides were synthesized by a modification ofthis method, using appropriate 2′-modified amidites such as thoseavailable from Glen Research, Inc., Sterling, Va. 2′-fluorooligonucleotides were synthesized as described in Kawasaki et al., J.Med. Chem. 1993, 36, 831. Briefly, the protected nucleosideN⁶-benzoyl-2′-deoxy-2′-fluoroadenosine was synthesized utilizingcommercially available 9-8-D-arabinofuranosyladenine as startingmaterial and by modifying literature procedures whereby the 2′-″-fluoroatom is introduced by a S_(N)2-displacement of a 2′-8-O-trifyl group.Thus N⁶-benzoyl-9-8-D-arabinofuranosyladenine was selectively protectedin moderate yield as the 3′,5′-ditetrahydropyranyl (THP) intermediate.Deprotection of the THP and N⁶-benzoyl groups was accomplished usingstandard methodologies and standard methods were used to obtain the5′-dimethoxytrityl-(DMT) and 5′-DMT-3′-phosphoramidite intermediates.

[0058] The synthesis of 2′-deoxy-2′-fluoroguanosine was accomplishedusing tetraisopropyldisiloxanyl (TPDS) protected9-8-D-arabinofuranosylguanine as starting material, and conversion tothe intermediate diisobutyryl-arabinofuranosylguanosine. Deprotection ofthe TPDS group was followed by protection of the hydroxyl group with THPto give diisobutyryl di-THP protected arabinofuranosylguanine. SelectiveO-deacylation and triflation was followed by treatment of the crudeproduct with fluoride, then deprotection of the THP groups. Standardmethodologies were used to obtain the 5′-DMT- and5′-DMT-3′-phosphoramidites.

[0059] Synthesis of 2′-deoxy-2′-fluorouridine was accomplished by themodification of a literature procedure in which2,2′-anhydro-1-8-D-arabinofuranosyluracil was treated with 70% hydrogenfluoride-pyridine. Standard procedures were used to obtain the 5′-DMTand 5′-DMT-3′phosphoramidites. 2′-deoxy-2′-fluorocytidine wassynthesized via amination of 2′-deoxy-2′-fluorouridine, followed byselective protection to give N⁴-benzoyl-2′-deoxy-2′-fluorocytidine.Standard procedures were used to obtain the 5′-DMT and5′-DMT-3′phosphoramidites.

[0060] Oligonucleotides having methylene(methylimino) backbones aresynthesized according to U.S. Pat. No. 5,378,825, which is coassigned tothe assignee of the present invention and is incorporated herein in itsentirety. Other nitrogen-containing backbones are synthesized accordingto WO 92/20823 which is also coassigned to the assignee of the presentinvention and incorporated herein in its entirety.

[0061] Oligonucleotides having amide backbones are synthesized accordingto De Mesmaeker et al., Acc. Chem. Res. 1995, 28, 366. The amide moietyis readily accessible by simple and well-known synthetic methods and iscompatible with the conditions required for solid phase synthesis ofoligonucleotides.

[0062] Oligonucleotides with morpholino backbones are synthesizedaccording to U.S. Pat. No. 5,034,506 (Summerton and Weller).

[0063] Peptide-nucleic acid (PNA) oligomers are synthesized according toP. E. Nielsen et al., Science 1991, 254, 1497).

[0064] After cleavage from the controlled pore glass column (AppliedBiosystems) and deblocking in concentrated ammonium hydroxide at 55 ECfor 18 hours, the oligonucleotides are purified by precipitation twiceout of 0.5 M NaCl with 2.5 volumes ethanol. Synthesized oligonucleotideswere analyzed by polyacrylamide gel electrophoresis on denaturing gelsand judged to be at least 85% full length material. The relative amountsof phosphorothioate and phosphodiester linkages obtained in synthesiswere periodically checked by ³¹p nuclear magnetic resonancespectroscopy, and for some studies oligonucleotides were purified byHPLC, as described by Chiang et al., J. Biol. Chem. 1991, 266, 18162.Results obtained with HPLC-purified material were similar to thoseobtained with non-HPLC purified material.

[0065] Oligonucleotides having 2′-O—CH₂CH₂OCH₃ modified nucleotides weresynthesized according to the method of Martin. Helv. Chim. Acta 1995,78, 486-504. All 2′-O—CH₂CH₂OCH³⁻cytosines were 5-methyl cytosines,synthesized as follows:

[0066] Monomers:

[0067] 2,2′-Anhydro[1-(β-D-arabinofuranosyl)-5-methyluridine]

[0068] 5-Methyluridine (ribosylthymine, commercially available throughYamasa, Choshi, Japan) (72.0 g, 0.279 M), diphenyl-carbonate (90.0 g,0.420 M) and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300mL). The mixture was heated to reflux, with stirring, allowing theevolved carbon dioxide gas to be released in a controlled manner. After1 hour, the slightly darkened solution was concentrated under reducedpressure. The resulting syrup was poured into diethylether (2.5 L), withstirring. The product formed a gum. The ether was decanted and theresidue was dissolved in a minimum amount of methanol (ca. 400 mL). Thesolution was poured into fresh ether (2.5 L) to yield a stiff gum. Theether was decanted and the gum was dried in a vacuum oven (60 EC at 1 mmHg for 24 h) to give a solid which was crushed to a light tan powder (57g, 85% crude yield). The material was used as is for further reactions.

[0069] 2′-O-Methoxyethyl-5-methyluridine

[0070] 2,2′-Anhydro-5-methyluridine (195 g, 0.81 M),tris(2-methoxyethyl)borate (231 g, 0.98 M) and 2-methoxyethanol (1.2 L)were added to a 2 L stainless steel pressure vessel and placed in apre-heated oil bath at 160 EC. After heating for 48 hours at 155-160 EC,the vessel was opened and the solution evaporated to dryness andtriturated with MeOH (200 mL). The residue was suspended in hot acetone(1 L). The insoluble salts were filtered, washed with acetone (150 mL)and the filtrate evaporated. The residue (280 g) was dissolved in CH₃CN(600 mL) and evaporated. A silica gel column (3 kg) was packed inCH₂Cl₂/acetone/MeOH (20:5:3) containing 0.5% Et₃NH. The residue wasdissolved in CH₂Cl₂ (250 mL) and adsorbed onto silica (150 g) prior toloading onto the column. The product was eluted with the packing solventto give 160 g (63%) of product.

[0071] 2!-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

[0072] 2′-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) wasco-evaporated with pyridine (250 mL) and the dried residue dissolved inpyridine (1.3 L). A first aliquot of dimethoxytrityl chloride (94.3 g,0.278 M) was added and the mixture stirred at room temperature for onehour. A second aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) wasadded and the reaction stirred for an additional one hour. Methanol (170mL) was then added to stop the reaction. HPLC showed the presence ofapproximately 70% product. The solvent was evaporated and trituratedwith CH₃CN (200 mL). The residue was dissolved in CHCl₃ (1.5 L) andextracted with 2×500 mL of saturated NaHCO₃ and 2×500 mL of saturatedNaCl. The organic phase was dried over Na₂SO₄ filtered and evaporated.275 g of residue was obtained. The residue was purified on a 3.5 kgsilica gel column, packed and eluted with EtOAc/Hexane/Acetone (5:5:1)containing 0.5% Et₃NH. The pure fractions were evaporated to give 164 gof product. Approximately 20 g additional was obtained from the impurefractions to give a total yield of 183 g (57%).

[0073]3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

[0074] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (106 g,0.167 M), DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL ofDMF and 188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M)were combined and stirred at room temperature for 24 hours. The reactionwas monitored by tlc by first quenching the tlc sample with the additionof MeOH. Upon completion of the reaction, as judged by tlc, MeOH (50 mL)was added and the mixture evaporated at 35 EC. The residue was dissolvedin CHCl₃ (800 mL) and extracted with 2×200 mL of saturated sodiumbicarbonate and 2×200 mL of saturated NaCl. The water layers were backextracted with 200 mL of CHCl₃. The combined organics were dried withsodium sulfate and evaporated to give 122 g of residue (approx. 90%product). The residue was purified on a 3.5 kg silica gel column andeluted using EtOAc/Hexane(4:1). Pure product fractions were evaporatedto yield 96 g (84%).

[0075]3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine

[0076] A first solution was prepared by dissolving3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (96g, 0.144 M) in CH₃CN (700 mL) and set aside. Triethylamine (189 mL, 1.44M) was added to a solution of triazole (90 g, 1.3 M) in CH₃CN (1 L),cooled to −5 EC and stirred for 0.5 h using an overhead stirrer. POCl₃was added dropwise, over a 30 minute period, to the stirred solutionmaintained at 0-10 EC, and the resulting mixture stirred for anadditional 2 hours. The first solution was added dropwise, over a 45minute period, to the later solution. The resulting reaction mixture wasstored overnight in a cold room. Salts were filtered from the reactionmixture and the solution was evaporated. The residue was dissolved inEtOAc (1 L) and the insoluble solids were removed by filtration. Thefiltrate was washed with 1×300 mL of NaHCO₃ and 2×300 mL of saturatedNaCl, dried over sodium sulfate and evaporated. The residue wastriturated with EtOAc to give the title compound.

[0077] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

[0078] A solution of3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine(103 g, 0.141 M) in dioxane (500 mL) and NH₄OH (30 mL) was stirred atroom temperature for 2 hours. The dioxane solution was evaporated andthe residue azeotroped with MeOH (2×200 mL). The residue was dissolvedin MeOH (300 mL) and transferred to a 2 liter stainless steel pressurevessel. MeOH (400 mL) saturated with NH₃ gas was added and the vesselheated to 100 EC for 2 hours (tic showed complete conversion). Thevessel contents were evaporated to dryness and the residue was dissolvedin EtOAc (500 mL) and washed once with saturated NaCl (200 mL). Theorganics were dried over sodium sulfate and the solvent was evaporatedto give 85 g (95%) of the title compound.

[0079]N⁴-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

[0080] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (85 g,0.134 M) was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g,0.165 M) was added with stirring. After stirring for 3 hours, tic showedthe reaction to be approximately 95% complete. The solvent wasevaporated and the residue azeotroped with MeOH (200 mL). The residuewas dissolved in CHCl₃ (700 mL) and extracted with saturated NaHCO₃(2×300 mL) and saturated NaCl (2×300 mL), dried over MgSO₄ andevaporated to give a residue (96 g). The residue was chromatographed ona 1.5 kg silica column using EtOAc/Hexane (1:1) containing 0.5% Et₃NH asthe eluting solvent. The pure product fractions were evaporated to give90 g (90%) of the title compound.

[0081]N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine-3′-amidite

[0082]N⁴-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (74g, 0.10 M) was dissolved in CH₂Cl₂ (1 L). Tetrazole diisopropylamine(7.1 g) and 2-cyanoethoxy-tetra-(isopropyl)phosphite (40.5 mL, 0.123 M)were added with stirring, under a nitrogen atmosphere. The resultingmixture was stirred for 20 hours at room temperature (tlc showed thereaction to be 95% complete). The reaction mixture was extracted withsaturated NaHCO₃ (1×300 mL) and saturated NaCl (3×300 mL). The aqueouswashes were back-extracted with CH₂Cl₂ (300 mL), and the extracts werecombined, dried over MgSO₄ and concentrated. The residue obtained waschromatographed on a 1.5 kg silica column using EtOAc\Hexane (3:1) asthe eluting solvent. The pure fractions were combined to give 90.6 g(87%) of the title compound.

[0083] 5-methylcytidine DMT β-cyanoethyl phosphoramidites arecommercially available from PerSeptive Biosystems (Framingham, Mass.).

Example 2 Evaluation of Inhibitory Activity of AntisenseOligonucleotides Which are Targeted to the Polyprotein TranslationInitiation Codon Region and Adjacent Core Protein Coding Region

[0084] (1) In order to evaluate the inhibitory activity of antisenseoligonucleotides which are complementary to the region including thetranslation initiation codon (nucleotide number 342-344) of HCV-RNA andthe adjacent core protein coding region, a series of 20 mer antisenseoligonucleotides were prepared which are complementary to the regionfrom nucleotide 320 to nucleotide 379. These are named according totheir target sequence on the HCV RNA, i.e., the oligonucleotide name(e.g., 330) is the number of the 5′-most nucleotide of the correspondingHCV RNA target sequence shown in SEQ ID NO: 37. Accordingly,oligonucleotide 330 is targeted to nucleotides 330-349 of the HCV RNAshown in SEQ ID NO: 37. Of these oligonucleotides, oligonucleotides 324through 344 contain all or part of the sequence CAT which iscomplementary to the AUG initiation codon itself. The nucleotidesequence of these antisense oligonucleotides are shown in Table 1. TABLE1 Antisense oligonucleotides to HCV % SEQ ID Oligo Sequence InhibitionNO: 320 TGC ACG GTC TAC GAG ACC TC 3 1 322 GGT GCA CGG TCT ACG AGA CC 52 324 ATG GTG CAC GGT CTA CGA GA 31 3 326 TCA TGG TGC ACG GTC TAC GA 394 328 GCT CAT GGT GCA CGG TCT AC 71 5 330 GTG CTC ATG GTG CAC GGT CT 386 332 TCG TGC TCA TGG TGC ACG GT 5 7 334 ATT CGT GCT CAT GGT GCA CG 39 8336 GGA TTC GTG CTC ATG GTG CA 98 9 338 TAG GAT TCG TGC TCA TGG TG 99 10340 TTT AGG ATT CGT GCT CAT GG 97 11 342 GGT TTA GGA TTC GTG CTC AT 9612 344 GAG GTT TAG GAT TCG TGC TC 99 13 344-i1 GAG GTT TAG GAT TIG TGCTC 95 14 344-i3 GIG GTT TIG GAT TIG TGC TC 90 15 344-i5 GIG GTT TIG GAIIIG TGC TC 51 16 346 TTG AGG TTT AGG ATT CGT GC 98 17 348 CTT TGA GGTTTA GGA TTC GT 98 18 350 TTC TTT GAG GTT TAG GAT TC 99 19 352 TTT TCTTTG AGG TTT AGG AT 99 20 354 GTT TTT CTT TGA GGT TTA GG 91 21 356 TGGTTT TTC TTT GAG GTT TA 86 22 358 TTT GGT TTT TCT TTG AGG TT 83 23 360CGT TTG GTT TTT CTT TGA GG 81 24

[0085] The inhibitory activity of these 21 antisense oligonucleotideswas evaluated in the in vitro translation assay. As shown in Table 1,antisense oligonucleotides 328, 336, 338, 340, 342, 344, 346, 348, 350,352, 354, 356, 358 and 360 showed an inhibitory activity of greater than70%, and are preferred. Of these, 336, 338, 340, 342, 344, 346, 348, 350and 352 showed an extremely high inhibitory activity of over 95% and aremost preferred.

[0086] The HCV target sequence regions complementary to the above 9 mostactive antisense oligonucleotides have in common the four nucleotidesfrom number 352 to 355 in the core protein coding region near thepolyprotein translation initiation codon. Thus, it is preferred totarget these four nucleotides in order to inhibit the translation.Accordingly, oligonucleotides comprising the sequence GGAT are preferredembodiments of the invention.

[0087] (2) Evaluation of antisense oligonucleotides in which thenucleotides known to be variable among strains were replaced by inosine:

[0088] It is known that in the nucleotide sequences in the core proteincoding region near the translation initiation codon, variation of basesamong strains occasionally occurs at nucleotides 350, 351, 352, 356 and362. Based on this knowledge, it was studied whether substitution ofthese bases by the “universal base” inosine would be effective forinhibition of various viruses.

[0089] An antisense DNA, designated oligonucleotide 344-il, was preparedin which the base at base number 350 in oligonucleotide 344 was replacedby inosine. Likewise, an antisense DNA, designated oligonucleotide344-i3, in which three bases at base numbers 350, 356 and 362 weresubstituted by inosine, and an antisense DNA, designated oligonucleotide344-i5, in which five bases at base numbers 350, 351, 352, 356, and 362were substituted by inosine, were prepared. The inhibitory activity ofthese antisense oligonucleotides was evaluated in the in vitrotranslation assay. As a result, oligonucleotides 344-il and 344-i3showed high inhibitory activity. Therefore, antisense oligonucleotidestargeted to nucleotides 344-363 of HCV RNA and which have three inosinesubstituents or less are preferred. Their inhibitory activities areshown in Table 1.

Example 3 Evaluation of Oligonucleotides 120, 330 and 340 and TruncatedVersions of Oligonucleotides 120, 260, 330 and 340 in H8Ad17 Cell Assayfor Effects on HCV RNA Levels

[0090] The anti-HCV activity of P═S oligonucleotides 120, 330 and 340was evaluated in H8Ad17 cells as follows.

[0091] An expression plasmid containing a gene (1.3 kb) coding for 5′NCR-core-env region of HCV gene was prepared by conventional methods andtransfected into a liver cell strain (H8Ad17) by lipofection accordingto standard methods. The desired liver cell transformant, whichexpressed HCV core protein, was obtained.

[0092] HCV RNA was isolated and quantitated by Northern blot analysis todetermine levels of expression. Core protein expression could also bedetected by ELISA method using an anti-HCV core-mouse monoclonalantibody as the solid phase antibody; an anti-HCV human polyclonalantibody as the primary antibody; and an HRP (horseradishperoxidase)-conjugated anti-human IgG-mouse monoclonal antibody as thesecondary antibody.

[0093] The liver cell transformant (2.5×10⁵ cells) were inoculated on6-well plates. To each plate was added each of the above-obtained fiveantisense oligonucleotides (each at a concentration of 5 μM). After twodays, the cells were harvested and counted. The cells were washed onceand lysed, and the inhibitory activity was measured by Northern blot.The inhibitory activities of the P═S antisense oligonucleotides werecalculated, compared to control without antisense oligonucleotide.

[0094] As before, the oligonucleotide number is the number of the5′-most nucleotide of the corresponding HCV RNA target sequence shown inSEQ ID NO: 37. For example, oligonucleotide 120 is a 20 mer targeted tonucleotides 120-139 of HCV RNA. Each of these compounds inducedreduction in HCV RNA levels at doses of 0.5 AM and 0.17 AM. These threecompounds (P═S 20 mers 120, 330 and 340) are therefore highly preferred.15 mer versions (truncated at by 5 nucleotides at either the 3′ or 5′end) induced a reduction of HCV RNA at the 0.5 AM dose. These compoundsare therefore preferred. 10 mers did not show sequence-specificinhibition at either dose.

[0095] A number of shortened analogs of oligonucleotide 330 were alsosynthesized as phosphorothioates and evaluated for effects on HCV RNAlevels in the same manner. The sequence of oligonucleotide 330 wastruncated at one or both ends. These oligonucleotides are shown in Table2. Oligonucleotide concentration was 100 nM. TABLE 2 Activity % SEQ IDOligo Sequence control NO 330 GTG CTC ATG GTG CAC GGT CT  30% 6 9559 GTGCTC ATG GTG CAC GGT 53 25 9557 GTG CTC ATG GTG CAC GG 52 26 9558 GTG CTCATG GTG CAC G 66 27 9036 GTG CTC ATG GTG CAC 37 28 9035 GTG CTC ATG G100  29 10471   G CTC ATG GTG CAC GGT CT 27 30 10470     CTC ATG GTG CACGGT CT 35 31 9033       C ATG GTG CAC GGT CT 32 32 9034              TGCAC GGT CT 82 33 10549  TG CTC ATG GTG CAC GGT C 17 34 10550   G CTC ATGGTG CAC GGT 36 35

[0096] In this assay, oligonucleotides 9036, 10471, 10470, 9038, 10549and 10550 gave greater than 50% inhibition of HCV RNA expression and aretherefore preferred.

Example 4 Evaluation of Oligos 259, 260 and 330 in the HCV H8Ad17 RNAAssay

[0097] The anti-HCV activity of P═S and 2′-O-propyl/P═S gappedoligonucleotides was evaluated in H8Ad17 cells as described in Example3. P═S oligonucleotides 259, 260 and 330 all induced similar (approx55%) reduction in HCV RNA levels in this assay, using 170 nMoligonucleotide concentration. The 2′-O-propyl gapped version ofoligonucleotide 259 showed approximately 25% inhibition of HCV RNAlevels (170 nM oligo dose), but oligonucleotides 260 and 330 were notactive as 2′-O-propyl gapped oligonucleotides in this assay. In aprevious assay of the same type, the gapped 2′-O-propyl version ofoligonucleotide 330 did induce a reduction of HCV RNA, though less thanwas observed for the P═S 330 oligonucleotide.

Example 5 Evaluation of oligos 259, 260 and 330 in an HCV H8Ad7 ProteinAssay

[0098] A Western blot assay employing affinity-purified human polyclonalanti-HCV serum and ¹²⁵1-conjugated goat anti-human IgG was developed inplace of ELISA assays previously used to evaluate effects ofoligonucleotides on HCV core protein levels. Six-well plates were seededwith H8 cells at 3.5×10⁵ cells/well. Cells were grown overnight. Cellswere treated with oligonucleotide in Optimem containing 5 μg/mllipofectin for 4 hours. Cells were fed with 2 ml H8 medium and allowedto recover overnight. To harvest cells, cells were washed once with 2 mlPBS, lysed in 100 μl Laemmli buffer and harvested by scraping. Forelectrophoresis, cell lysates were boiled, and 10-14 μl of cell lysatewas loaded on each lane of a 16% polyacrylamide gel. Afterelectrophoresing, proteins were transferred electrophoretically ontoPVDF membrane. The membrane was blocked in PBS containing 2% goat serumand 0.3% TWEEN-20, and incubated overnight with primary antibody (humananti-core antibody 2243 and rabbit anti-G3PDH antibody). The membranewas washed 5×5 minutes in buffer, then incubated with secondaryantibodies for 4-8 hours (125I-conjugated goat anti-human, and¹²⁵I-conjugated goat anti-rabbit). The membrane was washed 5×5 minutesin buffer, sealed in plastic and exposed in a PhosphorImager cassetteovernight. Bands were quantitated on the PhosphorImager (MolecularDynamics, Sunnyvale Calif.), normalized to G3PDH expression levels, andresults were plotted as a percentage of control untreated cells.

[0099] P═S and 2′-modified 330 oligonucleotides were evaluated usingthis Western blot assay. These oligonucleotides are shown in Table 3. Inthe sequences shown, capital letters represent base sequence, smallletters (o or s) represent internucleoside linkage, eitherphosphodiester (P═O) or phosphorothioate (P═S), respectively.Bold=2′-O-propyl. *=2′-O-butylimidazole. +=2′-O-propylamine. TABLE 3 SEQID Oligo # Sequence NO 330 GsTsGsCsTsCsAsTsGsGsTsGsCsAsCsGsGsTsCsT 6 330GsTsGsCsTsCsAsTsGsGsTsGsCsAsCsGsGsTsCsT 6* *                               * * 330GsTsGsCsTsCsAsTsGsGsTsGsCsAsCsGsGsTsCsT 6+ +                               + + 330GsTsGsCsTsCsAsTsGsGsTsGsCSAsCsGsGsTsCsT 6

[0100] Cells were treated with oligonucleotide at doses of 25 nM, 100 nMor 400 nM. The greatest reduction in core protein (approx 90-95% athigher doses) was observed with the P═S oligonucleotide. This compoundis therefore highly preferred. The 2′-O-propyl gapped P═Soligonucleotide gave approximately 80% inhibition of core proteinexpression. This compound is therefore preferred. The 2′-O-propyl/P═Ocompound did not show activity in this assay.

Example 6 Evaluation of Modified 330 Oligos in Cellular Assays

[0101] Oligonucleotides with the 330 sequence (SEQ ID NO: 6) andcontaining various modifications [P═S deoxy; 2′-O-propyl (uniform2′-O-propyl or 2′-O-propyl gapped, both uniformly P═S); or 2′-fluoromodifications (gapped or uniform, both uniformly P═S)] were evaluated inthe H8Ad17 core protein Western blot assay compared to a scrambledphosphorothioate control.

[0102] In this assay, the P═S oligonucleotide was consistently the best,giving an average of 62.4% inhibition of HCV core protein translation(n=7) compared to control. 2′-O-propyl and 2′-fluoro gappedoligonucleotides gave over 50% inhibition in at least one experiment.Uniformly 2′-fluoro or uniformly 2′-O-propyl oligonucleotides wereinactive in this experiment.

[0103] In this assay, the P═S oligonucleotides were consistently thebest and are preferred. Of these, P═S oligonucleotides 260, 270, 275,277 and 330 are more preferred. Uniform 2′fluoro P═S oligonucleotides345, 347 and 355 are also more preferred.

[0104] Additional uniform 2′-fluoro phosphorothioate oligonucleotideswere synthesized and tested for ability to inhibit HCV core proteinexpression. Oligonucleotide 344 was also found to be extremely activeand is preferred. The region of the HCV RNA target from nucleotide 344to nucleotide 374 was found to be extremely sensitive to antisenseoligonucleotide inhibition. Oligonucleotides complementary to thistarget region, therefore, are preferred. More preferred among these arethe 2′fluoro phosphorothioate oligonucleotides.

Example 7 Evaluation of a “Single Virus” Recombinant Vaccinia/HCV CoreProtein Assay

[0105] A “single virus”! vaccinia assay system was developed, which doesnot require co-infection with helper vaccinia virus expressing T7polymerase. Cells were pretreated with oligonucleotide in the absence oflipofectin prior to infection with recombinant vaccinia virus expressingHCV sequences. Cells were then infected with recombinant vaccinia virusexpressing HCV 5′ UTR-core at a m.o.i. of 2.0 pfu/cell. After infection,cells were rinsed and post-treated with medium containingoligonucleotide. Initial results obtained with this assay indicate thatP═S oligonucleotides 259 and 260 inhibit HCV 5′-UTR core expressionby >60% at a concentration of 1 μM. Inhibition is dose-dependent.

[0106] Uniformly 2′-fluoro P═S oligonucleotides 260, 330 and 340 wereevaluated for activity in the recombinant vaccinia “single virus” assayusing RY5 cells. Medium containing oligonucleotide was added afterinfection. 2′-fluoro modified oligonucleotide 260 induced adose-dependent inhibitory effect on HCV core protein expression (up toapproximately 65% inhibition) even without pretreatment of cells witholigonucleotide before infection. In the same assay with pretreatment,2′-fluoro P═S modified oligonucleotide 340 effectively inhibited HCVcore protein expression at doses of 0.1 μM, 0.3 μM and 1.0 μM, with amaximum inhibition of about 75%. This oligonucleotide is thereforepreferred. In the “single virus” assay using HepG2 cells, adose-dependent inhibitory effect of oligonucleotide 340 as a uniform2′-fluoro phosphorothioate was also observed (approximately 60%inhibition). This oligonucleotide is therefore preferred. Thephosphorothioate oligonucleotide 260 also gave approximately 60%inhibition in the HepG2 cell assay.

Example 8 Diagnostic Use of Oligonucleotides Which Inhibit HCV

[0107] Definitive diagnosis of HCV-caused hepatitis can be readilyaccomplished using antisense oligonucleotides which inhibit HCV RNAfunction, measurable as a decrease in HCV RNA levels or HCV core proteinlevels. RNA is extracted from blood samples or liver tissue samplesobtained by needle biopsy, and electrophoresed and transferred tonitrocellulose for Northern blotting according to standard methodsroutinely used by those skilled in the art. An identical sample of bloodor tissue is treated with antisense oligonucleotide prior to RNAextraction. The intensity of putative HCV signal in the two blots isthen compared. If HCV is present (and presumably causative of disease),the HCV RNA signal will be reduced in the oligonucleotide-treated samplecompared to the untreated sample. If HCV is not the cause of thedisease, the two samples will have identical signals. Similar assays canbe designed which employ other methods such as RT-PCR for HCV RNAdetection and quantitation, or Western blotting or ELISA measurement ofHCV core protein translation, all of which are routinely performed bythose in the art.

[0108] Diagnostic methods using antisense oligonucleotides capable ofinhibiting HCV RNA function are also useful for determining whether agiven virus isolated from a patient with hepatitis will respond totreatment, before such treatment is initiated. RNA is isolated from apatient's blood or a liver tissue sample and blotted as described above.An identical sample of blood or tissue is treated with antisenseoligonucleotide to inhibit HCV prior to RNA extraction and blotting. Theintensity of putative HCV signal in the two blots is then compared. Ifthe oligonucleotide is capable of inhibiting RNA function of thepatient-derived virus, the HCV signal will be reduced in theoligonucleotide-treated sample compared to the untreated sample. Thisindicates that the patient's HCV infection is responsive to treatmentwith the antisense oligonucleotide, and a course of therapeutictreatment can be initiated. If the two samples have identical signalsthe oligonucleotide is not able to inhibit replication of the virus, andanother method of treatment is indicated. Similar assays can be designedwhich employ other methods such as RT-PCR for RNA detection andquantitation, or Western blotting or ELISA for quantitation of HCV coreprotein expression, all of which are routinely performed by those in theart.

Example 9 The VHCV-IRES Vaccinia/HCV Recombinant Virus Infected MouseModel

[0109] pSC11 (licensed from N1H) is a vaccinia virus expression vectorthat uses vaccinia early and late promotor P7.5 to express foreigngenes, and vaccinia late promotor P11 to express a LacZ gene. Thevaccinia viral thymidine kinase (TK) sequence flanked these twopromoter-expression DNA arrangements for homologous recombination. HCVRNA nucleotides 1-1357, including the HCV 5′ noncoding region, core andpart of E1, obtained from pHCV3, a cDNA clone from a chronic HCV patientwith HCV type H infection, was fused to the 5′ end of a luciferase genecontaining a SV40 polyadenylation signal sequence (Promega, pGL-2promoter vector). The fused DNA fragment was placed under vacciniapromoter P7.5 of pSC11. The resultant construct was named pVNCELUA. Adeletion of HCV RNA nucleotides 709 to 1357 was made in pVNCELUA andreligation yielded the construct pVHCV-IRES. This construct uses the HCVinitiator with the internal ribosome entry initiating mechanism fortranslation. pVC-LUA is a luciferase control virus construct in whichthe luciferase gene including the translation initiation codon andpolyadenylation signal was directly placed under the P71.5 promoter ofpSC11.

[0110] The basic experimental procedures for generating recombinantvaccinia virus by homologous recombination are known in the art. CV-1cells for homologous recombination and viral plaque and Hu TK-143B forTK-selection were purchased from the ATCC. Plasmid DNA transfection wasdone using lipofectin (GIBCO BRL). The selection of recombinant viruswas done by selection of viral plaques resistant to BrdU anddemonstrating luciferase and β-galactosidase activity. The virus waspurified through three rounds of plaque selection and used to prepare a100% pure viral stock. The virus-containing BSC-40 cells were harvestedin DMEM with 0.5% FBS followed by freeze-thawing three times todissociate the virus. Cellular debris was centrifuged out and thesupernatant was used for viral infection. A capital “V” was given to thename of each recombinant virus to distinguish it from the correspondingDNA construct (named with “p”).

[0111] Six-week old female Balb/c mice were purchased from Charles RiverLaboratories (Boston Mass.). The mice were randomly grouped and werepretreated with oligonucleotide given subcutaneously once daily for twodays before virus infection and post-treated once at 4 hours afterinfection. The infection was carried out by intraperitoneal injection of1×10⁸ pfu of virus in 0.5 ml saline solution. At 24 hours afterinfection the liver was taken from each mouse and kept on dry ice untilit was homogenized at 30,000 rpm for about 30 seconds in 20 μl/mgluciferase reporter lysis buffer (Promega) using a Tissue Tearor(Biospec Products Inc.). Samples were transferred to eppendorf tubes onice and shaken by vortex for 20 seconds followed by centrifuging at 4°C. for 3 minutes 20 μl of supernatant was transferred to a 96-wellmicrotiter plate and 100 μl Luciferase Assay Reagent (Promega) wasadded. Immediately thereafter, the relative light units emitted weremeasured using a luminometer (ML 1000/Model 2.4, Dynatech Laboratories,Inc.).

Example 10 Evaluation of the 330 Oligonucleotide ISIS 6547 in theVCHV-IRES Infected Mouse Model:

[0112] A 20 mer deoxy oligonucleotide (the “330 oligonucleotide,” SEQ IDNO: 6) targeted to nucleotides 330-349 surrounding the HCV translationinitiation codon has been shown in previous examples to specificallyinhibit HCV core protein synthesis in an in vitro translation assay,when tested as a phosphodiester. The phosphorothioatedeoxyoligonucleotide of the same sequence, ISIS 6547 demonstrated atleast a 50% reduction of HCV RNA when administered at dose of 100 nM totransformed human hepatocytes expressing HCV 5′ noncoding region, core,and part of the E1 product (nucleotides 1-1357 of HCV). Hanecak et al.,J. Virol. 1996, 70, 5203-5212. This effect was dose-dependent andsequence-dependent.

[0113] ISIS 6547 was evaluated in vivo using the VHCV-IRES infectedmouse model. Eight female Balb/c mice were pretreated subcutaneouslywith oligonucleotide in saline once daily for two days, then infectedintraperitoneally with 1×10⁸ pfu VHCV-IRES followed by a post-treatmentwith oligonucleotide four hours after infection. A group treated withsaline and infected with the same amount of VHCV-IRES served ascontrols. The effect of oligonucleotide on HCV gene expression wasmeasured by luciferase activity at 24 hours after infection. Whencompared to luciferase activity from VHCV-IRES-infected butsaline-treated controls, ISIS 6547 reduced luciferase signal in adose-dependent manner, giving 10.5% inhibition at 2 mg/kg, 28.2%inhibition at 6 mg/kg and 51.9% inhibition at 20 mg/kg. In contrast, theunrelated control oligonucleotide ISIS 1082 (GCCGAGGTCCATGTCGTACGC; SEQID NO. 36) exhibited no inhibitory effect at lower doses, thoughnon-specific inhibition of luciferase signal was observed at 20 mg/kg.Various routes of administration of oligonucleotide 6547 (subcutaneous,intravenous or intraperitoneal) gave similar levels of inhibition (76%,63% and 58%, respectively, at 20 mg/kg).

Example 11 Evaluation of 5-methyl-C Modified 330 Oligonucleotide, ISIS14803 in the VCHV-IRES Infected Mouse Model

[0114] One of the heterocyclic base modifications presently available is5-methylcytosine (5-me-C) in which the nucleobase cytosine is methylatedat the 5-position. The corresponding nucleotide is 5-methylcytidine.Oligonucleotides containing this modification demonstrate higher targetbinding affinity than analogs without the base modification, and aresubstrates for RNAse H. Dean and Griffey, Antisense and Nucleic AcidDrug Development 1997, 7, 229-233. They also elicit less immunestimulation and complement stimulation than unmodified versions. Henryet al., Anti-Cancer Drug Design 1997, 12, 409-412.

[0115] A 5-me-C version of ISIS 6547 was synthesized in which everycytidine nucleotide was replaced by a 5-methylcytidine. Thisoligonucleotide, ISIS 14803, was evaluated in the VHCV-IRES system inmice, in direct comparison to its parent compound, ISIS 6547. Eightfemale Balb/c mice were subcutaneously treated with oligonucleotide insaline at one day and two hours before infection and again at 4 hoursafter infection. Mice were infected by intraperitoneal injection with1×10⁸ pfu per mouse of VHCV-IRES or VC-LUA. At 24 hours after infection,luciferase activity in liver was determined and compared to luciferaseactivity in livers of a group of mice treated with saline and infectedwith the same amount of VHCV-IRES or VC-LUA. ISIS 14803 showed 11.1%inhibition of liver luciferase activity at 2 mg/kg, 33.5% inhibition at6 mg/kg and 59.1% inhibition at 20 mg/kg. ISIS 14803 did not show anyinhibition of luciferase activity in the control VC-LUA virus at thelower doses, though some nonspecific inhibition of luciferase activitywas observed at the high dose of 20 mg/kg. Because this nonspecificinhibition was also observed with the control oligonucleotide, ISIS1082, it was thought to be a general class effect of high doses ofphosphorothioate oligonucleotides.

1 37 1 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 1 tgcacggtct acgagacctc 20 2 20 DNA Artificial SequenceDescription of Artificial Sequence Synthetic 2 ggtgcacggt ctacgagacc 203 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 3 atggtgcacg gtctacgaga 20 4 20 DNA Artificial SequenceDescription of Artificial Sequence Synthetic 4 tcatggtgca cggtctacga 205 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 5 gctcatggtg cacggtctac 20 6 20 DNA Artificial SequenceDescription of Artificial Sequence Synthetic 6 gtgctcatgg tgcacggtct 207 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 7 tcgtgctcat ggtgcacggt 20 8 20 DNA Artificial SequenceDescription of Artificial Sequence Synthetic 8 attcgtgctc atggtgcacg 209 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 9 ggattcgtgc tcatggtgca 20 10 20 DNA Artificial SequenceDescription of Artificial Sequence Synthetic 10 taggattcgt gctcatggtg 2011 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 11 tttaggattc gtgctcatgg 20 12 20 DNA Artificial SequenceDescription of Artificial Sequence Synthetic 12 ggtttaggat tcgtgctcat 2013 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 13 gaggtttagg attcgtgctc 20 14 20 DNA Artificial SequenceDescription of Artificial Sequence Synthetic 14 gaggtttagg attngtgctc 2015 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 15 gnggtttngg attngtgctc 20 16 20 DNA Artificial SequenceDescription of Artificial Sequence Synthetic 16 gnggtttngg annngtgctc 2017 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 17 ttgaggttta ggattcgtgc 20 18 20 DNA Artificial SequenceDescription of Artificial Sequence Synthetic 18 ctttgaggtt taggattcgt 2019 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 19 ttctttgagg tttaggattc 20 20 20 DNA Artificial SequenceDescription of Artificial Sequence Synthetic 20 ttttctttga ggtttaggat 2021 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 21 gtttttcttt gaggtttagg 20 22 20 DNA Artificial SequenceDescription of Artificial Sequence Synthetic 22 tggtttttct ttgaggttta 2023 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 23 tttggttttt ctttgaggtt 20 24 20 DNA Artificial SequenceDescription of Artificial Sequence Synthetic 24 cgtttggttt ttctttgagg 2025 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 25 gtgctcatgg tgcacggt 18 26 17 DNA Artificial SequenceDescription of Artificial Sequence Synthetic 26 gtgctcatgg tgcacgg 17 2716 DNA Artificial Sequence Description of Artificial Sequence Synthetic27 gtgctcatgg tgcacg 16 28 15 DNA Artificial Sequence Description ofArtificial Sequence Synthetic 28 gtgctcatgg tgcac 15 29 10 DNAArtificial Sequence Description of Artificial Sequence Synthetic 29gtgctcatgg 10 30 18 DNA Artificial Sequence Description of ArtificialSequence Synthetic 30 gctcatggtg cacggtct 18 31 17 DNA ArtificialSequence Description of Artificial Sequence Synthetic 31 ctcatggtgcacggtct 17 32 15 DNA Artificial Sequence Description of ArtificialSequence Synthetic 32 catggtgcac ggtct 15 33 10 DNA Artificial SequenceDescription of Artificial Sequence Synthetic 33 tgcacggtct 10 34 18 DNAArtificial Sequence Description of Artificial Sequence Synthetic 34tgctcatggt gcacggtc 18 35 16 DNA Artificial Sequence Description ofArtificial Sequence Synthetic 35 gctcatggtg cacggt 16 36 21 DNAArtificial Sequence Description of Artificial Sequence Synthetic 36gccgaggtcc atgtcgtacg c 21 37 685 RNA Hepatitis C virus 37 gccagcccccgauugggggc gacacuccac cauagaucac uccccuguga ggaacuacug 60 ucuucacgcagaaagcgucu agccauggcg uuaguaugag ugucgugcag ccuccaggac 120 ccccccucccgggagagcca uaguggucug cggaaccggu gaguacaccg gaauugccag 180 gacgaccggguccuuucuug gaucaacccg ccaaugccug gagauuuggg cgugcccccg 240 cgagacugcuagccgaguag uguugggucg cgaaaggccu ugugguacug ccugauaggg 300 ugcuugcgagugccccggga ggucucguag accgugcacc augagcacga auccuaaacc 360 ucaaagaaaaaccaaacgua acaccaaccg ccgcccacag gaggucaagu ucccgggcgg 420 uggucagaucguugguggag uuuaccuguu gccgcgcagg ggccccaggu ugggugugcg 480 cgcgaucaggaagacuuccg agcggucgca accccgugga aggcgacagc cuauccccaa 540 ggcucgccggcccgagggca gggccugggc ucagcccggg uauccuuggc cccucuaugg 600 caaugagggcaugggguggg caggauggcu ccugucaccc cgcggcuccc ggccuaguug 660 gggccccacggacccccggc guagg 685

What is claimed is:
 1. An oligonucleotide 5 to 50 nucleotides in lengthwhich is complementary to at least a portion of an HCV genomic ormessenger RNA, said oligonucleotide being hybridizable to said RNA andcapable of inhibiting the function of said RNA.
 2. The oligonucleotideof claim 1 wherein said RNA comprises at least a portion of thepolyprotein translation initiation codon of an HCV RNA.
 3. Theoligonucleotide of claim 2 comprising SEQ ID NO:
 6. 4. A compositioncomprising the oligonucleotide of claim 1 in a pharmaceuticallyacceptable carrier.
 5. The composition of claim 4 further comprisinginterferon.
 6. A phosphorothioate oligodeoxynucleotide having SEQ ID NO:6 wherein every cytidine nucleotide is a 5-methylcytidine.
 7. Acomposition comprising the oligonucleotide of claim 6 in apharmaceutically acceptable carrier.
 8. The composition of claim 7further comprising interferon.
 9. A method for inhibiting the activityof a Hepatitis C virus comprising contacting the virus or cells infectedwith the virus with an effective amount of the oligonucleotide ofclaim
 1. 10. A method for inhibiting the activity of a Hepatitis C viruscomprising contacting the virus or cells infected with the virus with aneffective amount of the oligonucleotide of claim
 6. 11. A method fortreating an HCV-associated disease comprising contacting an animalsuspected of having an HCV-associated disease, or tissues, cells or abodily fluid from said animal, with a therapeutically effective amountof the oligonucleotide of claim
 1. 12. The method of claim 11 whereinthe HCV-associated disease is acute HCV infection, fulminant hepatitis,chronic active hepatitis, cirrhosis, or hepatocellular carcinoma.
 13. Amethod for treating an HCV-associated disease comprising contacting ananimal suspected of having an HCV-associated disease, or tissues, cellsor a bodily fluid from said animal, with a therapeutically effectiveamount of the oligonucleotide of claim
 6. 14. The method of claim 13wherein the HCV-associated disease is acute HCV infection, fulminanthepatitis, chronic active hepatitis, cirrhosis, or hepatocellularcarcinoma.
 15. A method for preventing an HCV-associated diseasecomprising contacting an animal suspected of having been exposed to HCV,or cells, tissues or a bodily fluid from said animal, with aprophylactically effective amount of the oligonucleotide of claim
 1. 16.The method of claim 15 wherein the HCV-associated disease is acute HCVinfection, fulminant hepatitis, chronic active hepatitis, cirrhosis, orhepatocellular carcinoma.
 17. A method for preventing an HCV-associateddisease comprising contacting an animal suspected of having been exposedto HCV, or cells, tissues or a bodily fluid from said animal, with aprophylactically effective amount of the oligonucleotide of claim
 6. 18.The method of claim 17 wherein the HCV-associated disease is acute HCVinfection, fulminant hepatitis, chronic active hepatitis, cirrhosis, orhepatocellular carcinoma.